Commercial efforts employing recombinant DNA technology for producing various polypeptides have, up to now, centered predominantly on Escherichia coli as a host organism. However, in some situations E. coli may prove to be unsuitable as a host. For example, E. coli contains a number of toxic pyrogenic factors that must be eliminated from any polypeptide useful as a pharmaceutical product. The efficiency with which this purification can be achieved will, of course, vary with the particular polypeptide. In addition, the polyteolytic activities of E. coli can seriously limit yields of some useful products. Moreover, proteins expressed at high levels in E. coli are packaged into refractory bodies and are difficult to solubilize. Thus recovery of active proteins from E. coli can frequently be troublesome. These and other considerations have led to increased interest in alternative hosts, in particular, the use of eukaryotic organisms for the production of polypeptide products is appealing.
The availability of means for the production of polypeptide products in eukaryotic systems, e.g., yeast, could provide significant advantages relative to the use of prokaryotic systems such as E. coli for the production of polypeptides encoded by recombinant DNA. Yeast has been employed in large scale frementations for centuries, as compared to the relatively recent advent of large scale E. coli fermentations. Yeast can generally be grown to higher cell densities than bacteria and are readily adaptable to continuous fermentation processing. In fact, growth of yeast such as Pichia pastoris to ultra-high cell densities, i.e., cell densities in excess of 100 g/L, is disclosed by Wegner in U.S. 4,414,329 (assigned to Phillips Petroleum Co.). Additional advantages of yeast hosts include the fact that many critical functions of the organism, e.g., oxidative phosphorylation, are located within organelles, and hence are not exposed to the possible deleterious effects of the organism's production of polypeptides foreign to the wild-type host cells. As a eukaryotic organism, yeast may prove capable of glycosylating expressed polypeptide products where such glycosylation is important to the bioactivity of the polypeptide product. It is also possible that as a eukaryotic organism, yeast will exhibit the same codon preferences as higher organisms, thus tending toward more efficient production of expression products from mammalian genes or from complementary DNA (cDNA) obtained by reverse transcription from, for example, mammalian mRNA.
The development of poorly characterized yeast species as host-vector systems is severely hampered by the lack of strains having auxotrophic mutations. Such mutant strains are often not available, precluding a direct selection for transformants by auxotrophic complementation. In order to avoid the need for auxotrophic mutants, it would be desirable to have available positive selection markers which would allow direct transformation of wild-type hosts. Wild-type hosts are desirable host organisms because they are readily available and require no strain manipulation in order to be useful as hosts to inserted DNA. Thus, dominant transformation markers which allow the direct transformation of prototrophic hosts are desirable.